“Apoptosis” refers to programmed cell death which occurs by an active, physiological process (Kerr, J. F., et al., 1972; Wyllie, A. H., 1980). Cells that die by apoptosis undergo characteristic morphological changes, including cell shrinkage and nuclear condensation and fragmentation. Apoptosis plays an important role in developmental processes, including morphogenesis, maturation of the immune system, and tissue homeostasis whereby cell numbers are limited in tissues that are continually renewed by cell division (Ellis, R. E., et al., 1991; Oppenheim, R. W., et al., 1991; Cohen, J. J., et al., 1992; Raff, M. C. 1992). Moreover, apoptosis is an important cellular safeguard against tumorigenesis (Williams, G. T., 1991; Lane, D. P., 1993). Defects in the apoptotic pathway causing disregulated or aberrant apoptosis may contribute to the onset or progression of malignancies. Under certain conditions, cells undergo apoptosis in response to the forced expression of oncogenes, or other genes that drive cell proliferation (Askew, D., et al., 1991; Evan, G. I., et al., 1992; Rao, L., et al., 1992; Smeyne, R. J., et al., 1993).
A variety of diseases and degenerative disorders may involve aberrant or disregulated apoptosis, resulting in inappropriate or premature cell death or inappropriate cell proliferation (Barr, P. J., et al., 1994). For example, inhibition of cell death may contribute to disease in the immune system by allowing the persistence of self-reactive B and T cells, which consequently promotes autoimmune disease (Watanabe-Fukunaga et al., 1992). Moreover, cancer may result when cells that fail to die undergo further mutations leading to a transformed state of the cells (Korsmeyer, S. J., 1992).
The productive infection by certain viruses may depend on suppression of host cell death by anti-apoptotic viral gene products (Rao, L., et al., 1992; Ray, C. A., et al., 1992; White, E., et al., 1992; Vaux, D. L., et al., 1994), and inhibition of apoptosis can alter the course (i.e., lytic vs. latent) of viral infection (Levine, B., et al., 1993). Moreover, the widespread apoptosis of T lymphocytes triggered by HIV infection may, at least in part, be responsible for the immune system failure associated with AIDS (Gougeon, M., et al., 1993). The roles of apoptosis in normal and pathological cell cycle events are reviewed in Holbrook, N. J. et al., 1996. Importantly, apoptosis comprises an important antiviral defense mechanism in animals and humans by providing the means to rapidly eliminate virally infected cells and restrict viral propagation (O'Brien, 1998; Tschopp et al., 1998). Apoptosis of virally infected cells is triggered by killer cells of the immune system via Fas-ligand interaction with Fas and by granzyme-B-triggered caspase activation (Nagata and Golstein, 1995; Smyth and Trapani, 1998).
To counteract the host defense mechanism, many viruses encode genes that function to inhibit or diminish apoptosis in infected cells (O'Brien, 1998; Tschopp et al., 1998). This inhibition or diminution of apoptosis by viral gene products is achieved by a variety of mechanisms, including: 1) blocking and/or destruction of p53; 2) direct interaction with cellular polypeptides of apoptotic pathways, such as death-effector-domain-containing polypeptides [death-effector-domain motifs are defined in Hu et al., 1997], Bcl-2 family members, and caspases; or 3) by induction of cellular anti-apoptotic polypeptides (Pilder et al., 1984; Gooding et al., 1988; Clem et al., 1991; Hershberger et al., 1992; Brooks et al., 1995; Sedger and McFadden, 1996; Leopardi and Roizman, 1996; Leopardi et al., 1997; Razvi and Welsh, 1995; Teodoro and Branton, 1997; Vaux et al., 1994; Shen and Shenk, 1995; Duke et al., 1996; Vaux and Strasser, 1996; Thompson, 1995).
The prevalence and evolutionary conservation of anti-apoptotic viral genes suggests that suppression of apoptosis is a critical component of efficient viral propagation and/or persistence in vivo. In fact, some of the anti-apoptotic genes were found to be essential for the ability of the respective viruses to replicate and propagate. For example, mutants of human adenovirus that lack the expression of the E1B 19 kDa adenoviral analog of Bcl-2 induce massive apoptosis of infected cells (Teodoro and Branton, 1997) which, consequently, leads to reduced viral titers.
Human cytomegalovirus (HCMV) is widespread in human populations, and is of substantial clinical importance principally because of its pathogenicity in developing fetuses and immunocompromised individuals (Huang and Kowalik, 1993; Britt and Alford, 1996). In particular, those immunocompromised individuals undergoing organ and tissue transplants, or that have malignancies and are receiving immunosuppressive chemotherapy, or that have AIDS, are at greatest risk of HCMV-induced diseases. These diseases range from developmental abnormalities, mental retardation, deafness, mononucleosis, and chorioretinitis, to fatal diseases like interstitial pneumonitis and disseminated HCMV infections (Huang and Kowalik, 1993; Britt and Alford, 1996).
Human cytomegalovirus (HCMV) is a herpesvirus (Roizman, 1991). A number of herpesviruses were shown to induce an apoptotic host cell response, and to suppress this virus-induced apoptosis in the infected cells (Leopardi and Roizman, 1996; Leopardi et al., 1997; Bertin et al., 1997; Sieg et al., 1996). The genomes of several herpesviruses code for a variety of anti-apoptotic polypeptides such as: 1) Bcl-2 homologs, e.g., BHRF-1 of Epstein-Barr virus (Henderson et al., 1993), vbcl-2 of Kaposi's sarcoma-associated herpesvirus (Sarid et al., 1997), and ORF16 of herpesvirus Saimiri (Nava et al., 1997); 2) a polypeptide that induces several cellular anti-apoptotic genes, e.g., LMP-1 of Epstein-Barr virus (Henderson et al., 1991; Wang et al., 1996; Fries et al., 1996); 3) a polypeptide interacting with FLICE (also called caspase 8), e.g., Equine herpesvirus type 2 polypeptide E8 (Bertin et al., 1997; Hu et al., 1997); and 4) two polypeptides with anti-apoptotic properties with a yet poorly characterized mechanism, ICP4 and US3 of HSV-1 (Leopardi and Roizman, 1996; Leopardi et al., 1997).
While there are several examples of anti-apoptotic genes encoded by other herpesviruses (Tschopp et al., 1998), little is known about the role of apoptosis in HCMV infections. It has been observed that HCMV-infected human cells acquire resistance towards apoptosis induced by serum withdrawal (Kovacs et al., 1996), and by infection of a mutant adenovirus which lacks the expression of the anti-apoptotic polypeptide E1B 19K (Zhu et al., 1995). Two immediate early polypeptides of HCMV, IE1 and IE2, were reported to each exhibit anti-apoptotic activity in some settings (Zhu et al., 1995). However, these viral polypeptides did not suppress apoptosis in other assays (see below).
The HCMV genome (AD169 strain) has been completely sequenced (Chee et al. 1990; Mocarski, 1996). The 230 kb HCMV genome is predicted to encode over 200 polypeptides, many of which have undefined functions (Chee et al., 1990; Mocarski et al., 1996), and none of which bears overt homology to known classes of cell death suppressors (e.g., the Bcl-2 family). Whether most of the HCMV genes are expressed and have any functional importance for HCMV replication remains unknown. In fact, a number of the predicted ORFs were found to be dispensable for the replication and/or propagation of HCMV in cultured cells (Mocarski, 1996).
Aside from IE1 and IE2, no other HCMV anti-apoptotic genes have been identified, and no homology to any of the known anti-apoptotic polypeptides has been found in the HCMV genome. Prior to the discoveries embodied herein, little was known about the UL36 and UL37 genes of HCMV. On the basis of DNA sequence analysis and RNA transcription studies, it was predicted that UL36 has two exons which encode a polypeptide product pUL36, and that UL37 encodes two polypeptide products; pUL37S encoded by the first exon (also called pUL37x1, see Tenney and Colberg-Poley, 1991 a), and pUL37L (also called gpUL37, see Zhang et al., 1996) encoded by all three exons (Chee et al., 1990; Tenney and Colberg-Poley, 1991a, b). The expression of pUL37L in HCMV-infected cells has been detected. However, prior to the discoveries embodied herein, it was not clear whether the hypothetical polypeptide pUL37S was expressed in HCMV-infected cells. Moreover, pUL37M had not yet been discovered.
Today, there are only very limited treatment options available for cytomegalovirus infections, and treatment is often associated with high toxicity and the generation of drug resistance (Hirsch, 1994; White and Fenner, 1994; Lalezari et al., 1997). Several potential drug targets for herpesviruses have recently been identified (White and Fenner, 1994, page 267, Table 16.1). Most of the antiviral compounds thus far developed, function to prevent or inhibit viral replication. For example, the currently available antiviral compounds, Gancyclovir, Foscarnet (PFA, phosphonoformic acid) and Cidofovir all act as inhibitors of viral DNA polymerase. There are no available antiviral compounds that function to inhibit or diminish viral infection or to eliminate virally infected cells by regulating or modulating the anti-apoptotic activity of viral polypeptides and/or the expression of viral genes or polynucleotides encoding polypeptides having anti-apoptotic activity, and thereby inducing or restoring apoptosis in virally infected cells.